Dependence of Nitrite Reduction on Electron Transport Chloroplasts

Abstract

Methyl viologen and phenazine methosulfate (photosystem I electron acceptors), 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU, electron-transport inhibitor), and methylamine (photophosphorylation uncoupler) were used to study the dependence of nitrite reduction on electron transport in chloroplasts.DCMU, methyl viologen, and phenazine methosulfate markedly inhibited, whereas methylamine stimulated NO2-reduction in isolated, intact spinach (Spinacia oleracea L.) chloroplasts. The objective of this study was to use isolated, intact chloroplasts and leaf sections to: (a) show that NO2 reduction is functionally associated with the electron-transport system of the chloroplasts; (b) seek additional evidence that Fd is the electron donor for NO2-reduction in leaves; and (c) evaluate the concept that ATP is not an obligatory metabolite in NO2-reduction. Chloroplast Preparation. The procedures for chloroplast preparation were as described (15), with the following exceptions. Isoascorbate was omitted from the grinding medium A as well as the resuspension medium B. After the homogenate was filtered to remove cell debris, the filtrate was centrifuged at 1SOOg for S min. The pellet was resuspended with 5 ml of medium B, and centrifuged as before. The pellet was again resuspended in 5 ml of medium B and used as a source of intact chloroplasts. Previous work (15) has shown that similar preparations contained approximately 50% intact (Class I) chloroplasts. MATERIALS AND METHODSNitrite Assay. A 2-ml aliquot of the chloroplast preparation was combined with 0.25 ml of 5 mm KNO2 and H20 plus additives (photosynthetic inhibitor, electron acceptors, or uncoupler) to make a final volume of 3.0 ml. The reaction mixtures were incubated in a water bath at 30 C with continuous shaking. Light was provided by a 500-w flood lamp which gave an incident energy of approximately 1.5 X 105 ergs cm-2 sec-1. Nitrite loss was followed by removing 0.2-ml aliquots of the reaction mixture at zero and subsequent time intervals. Nitrite 480 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

Nitrite Uptake into Intact Pea Chloroplasts

“…PMS was reported to catalyze cyclic electron flow in PSI, coupling the electron flow to ATP formation (29). The observed inhibition of nitrite uptake by PMS supported the proposal made by previous workers that, although PMS stimulates ATP formation (29), the availability of reductant for nitrogen assimilation is decreased (24). From present evidence it appeared that nitrite uptake was more closely dependent upon nitrite reduction than PSI phosphorylation.…”

“…A preliminary study was unable to differentiate nitrite uptake into intact chloroplasts from internal nitrite accumulation and reduction (3). Nitrite reduction is reported to be functionally associated with PSI electron transport, and not directly linked to ATP synthesis (24). It was, therefore, of interest to the present study to include the effect ofthe plastoquinone antagonist, DBMIB, on nitrite uptake.…”

“…In the present work, this problem was approached by employing electron transport (and therefore nitrite reduction [24]) regulators, uncouplers, and photophosphorylation inhibitors and, in addition, membrane penetrating and nonpenetrating protein binding reagents.…”

Effects of Certain Herbicides and Their Combinations on Nitrate and Nitrite Reduction

“…Nitrite reduction is inhibited by photosynthetic inhibitor herbicides (6,8,9,14) with little or no immediate effect upon nitrate reduction. In contrast, certain "nonphotosynthetic inhibitor" herbicides stimulate nitrate reduction and permit it to occur in darkness with little or no immediate effect upon photosynthetic nitrite reduction (10).…”

“…Nitrate reductase is located in the cytoplasm (16), uses NADH as its electron donor (2), and derives its source of NADH primarily from the oxidation of 3-P-glyceraldehyde (11) or malate (15). Nitrite reductase is located in the chloroplast (16), utilizes reduced ferredoxin as its electron donor (14), and derives its reductive energy from photosynthetic electron flow (14).…”